The centrosome serves as the major organizing center of the microtubule network in animal cells, which is used to support vesicle trafficking, establish and maintain cell polarity, organize cytoplasmic organelles, and evenly divide the chromosomes during mitosis. Defects in the organization of the microtubule network during mitosis usually result in unequal segregation of the chromosomes, resulting in aneuploidy, which can lead to a large number of catastrophic cell fates, including oncogenesis. Despite this significant role, relatively little is known about the protein composition and the interaction between proteins in the centrosome that allow it to carry out its diverse functions. This proposal seeks to utilize novel mass spectrometry based technologies to identify key proteins involved in the structure and regulation of the centrosome in a global manner. Double stranded RNA interference and metabolic stable isotope labeling will be used to quantify changes in centrosome protein composition in response to depletion of regulatory kinases in Drosophilla cells and early embryos. In addition, phosphorylation sites will be mapped and changes in the levels of phosphorylated kinase substrates will be quantified using this technique.